Biological methods for preparing adipic acid
The technology relates in part to biological methods for producing adipic acid and engineered microorganisms capable of such production.
1. A genetically modified Candida yeast, comprising:
a genetic modification that reduces or eliminates a PDX4 activity, thereby partially blocking beta oxidation activity;
a genetic modification that adds or increases a CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity; and
a genetic modification that adds or increases a monooxygenase reductase activity;
wherein said yeast is capable of producing adipic acid, at a level of at least 0.4 grams per liter of culture medium, from a feedstock comprising a mixture of fatty acids from a vegetable oil.
2. The genetically modified Candida yeast of claim 1 , wherein the Candida yeast is a genetically modified ATCC20336 yeast.
3. The genetically modified Candida yeast of claim 1 , wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A15 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A15 monooxygenase coding sequence.
4. The genetically modified Candida yeast of claim 1 , wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises: (i) an increased copy number of a CYP52A16 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A16 monooxygenase coding sequence.
5. The genetically modified Candida yeast of claim 1 , wherein the genetic modification that adds or increases the CYP52A15, CYP52A16 or CYP52A19 monooxygenase activity comprises (i) an increased copy number of a CYP52A19 monooxygenase coding sequence, or (ii) a modified or added promoter in operable linkage with a CYP52A19 monooxygenase coding sequence.
6. The genetically modified Candida yeast of claim 1 , wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRA monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRA monooxygenase reductase coding sequence.
7. The genetically modified Candida yeast of claim 1 , wherein the genetic modification that adds or increases the monooxygenase reductase activity comprises (i) an increased copy number of a CPRB monooxygenase reductase coding sequence, or (ii) a modified or added promoter in operable linkage with a CPRB monooxygenase reductase coding sequence.
8. The genetically modified Candida yeast of claim 1 , wherein the genetic modification that reduces or eliminates the PDX4 activity comprises (i) a disrupted, deleted or knocked out polynucleotide that encodes a PDX4 polypeptide, or (ii) a disrupted promoter and/or 5′UTR in functional connection with a polynucleotide that encodes a PDX4 polypeptide.
9. The genetically modified Candida yeast of claim 1 , wherein the vegetable oil is a palm oil, soybean oil or fatty acid soap stock.